Project Summary/Abstract The sexually transmitted human pathogens Neisseria gonorrhoeae and Chalmydia tracomatis each cause over 100 million infections worldwide each year. Since the 1940s individuals with gonorrhea have been cured of this disease by antibiotic therapy, which commonly includes treatment with antimicrobials against chlamydial co- infections. However, with the recent emergence of ceftriaxone-resistant (CroR) strains of N. gonorrhoeae, it is of great concern that in the absence of new antibiotics, gonorrhea may soon become an untreatable disease. The central hypothesis of this proposal is that antibiotic-resistant variants of N. gonorrhoeae evolve due to the selective pressure exerted by both antibiotic therapy and host antimicrobials that are produced during infection. We further posit that mutations in penicillin-binding protein 2 (PBP2), the lethal target of ceftriaxone, can negatively influence in vivo fitness, but that compensatory mutations develop to reverse this defect. To test these hypotheses, we will examine the fitness consequences for gonococci when they acquire resistance to cephalosporins. Ceftriaxone-resistant (CroR) gonococcal strains obtained from patients differ from susceptible strains in two ways: a) they contain a highly mosaic penA allele (e.g., penA41) encoding an extensively remodeled PBP2 that is poorly acylated by ceftriaxone, and b) they have regulatory mtr mutations that enhance expression of the MtrC-MtrD-MtrE efflux pump operon and confer resistance to host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs), and antibiotics, including beta-lactams. Our preliminary data reveal that strains harboring a mosaic penA have a fitness defect compared to wild-type strains both in vitro and in the female mouse model of gonococcal infection, thereby reducing their potential for persistence in the host and transmission in the community, but that they acquire mutations that increase their biological fitness, potentially mimicking what occurs in humans. In this proposal, we will: i) identify the mutation(s) in compensatory mutants of FA19 penA41 and elucidate the mechanisms by which these mutations increase fitness (Specific Aim 1); ii) define the role of mtr mutations in fitness of gonococci with a mosaic penA allele and identify the changes in transcription following acquisition of a mosaic penA allele (Specific Aim 2); and iii) examine if genes known to be important in gonococcal resistance to CAMPs (e.g. mtr or lptA) contribute to fitness advantages in CroR penA mutant strains, particularly under conditions (e.g. during a chlamydial co-infection) that decrease the production of CAMPs in the host (Specific Aim 3). We also will employ biochemical studies to define the alterations in peptidoglycan structure due to acquisition of a mosaic penA allele and to delineate whether the changes in peptidoglycan structure alter pro-inflammatory responses in host cells. Working closely with other members of the AC STI CRC, we will employ an experimental murine model of gonococcal infection, including co-infection with Chlamydia, to test the importance of the host response in the selection of CroR gonococci with enhanced fitness during infection. The results obtained from our work will advance our knowledge of the factors important in the emergence and spread of antibiotic resistant gonococci, as well as provide insights for the development of novel antimicrobials effective against such strains. Such studies are critical in this era when antibiotic resistance threatens the future of effective therapy against gonorrhea.